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Arthritis Res Ther. 2012; 14(3): R105.
Published online May 2, 2012. doi:  10.1186/ar3830
PMCID: PMC3446482
The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells
Akihiko Hiyama,corresponding author1,2 Fumiyuki Arai,1,2 Daisuke Sakai,1,2 Katsuya Yokoyama,1,2 and Joji Mochida1,2
1Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
2Research Center for Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
corresponding authorCorresponding author.
Akihiko Hiyama: a.hiyama/at/tokai-u.jp; Fumiyuki Arai: brabus/at/is.icc.u-tokai.ac.jp; Daisuke Sakai: daisakai/at/is.icc.u-tokai.ac.jp; Katsuya Yokoyama: katsuya/at/is.icc.u-tokai.ac.jp; Joji Mochida: jomo/at/is.icc.u-tokai.ac.jp
Received November 28, 2011; Accepted May 2, 2012.
Abstract
Introduction
The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.
Methods
Rat nucleus pulposus cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.
Results
Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.
Conclusions
These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.
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